This project will study fibroblast differentiation during specialization of connective tissues, concentrating especially on differentiation of keratocytes of the corneal stroma from the neural crest population of embryonic progenitor cells in the head region, versus differentiation of fibroblasts in other ocular and non-ocular tissues. Markers of keratocyte differentiation are ability to synthesize the three major keratan sulfate proteoglycan (KSPG) core proteins (lumican, keratocan, KSPG 25 or mimican) and glycosylate them with long, highly sulfated chains of keratan sulfate (KS). Such "KSylation" is a unique feature of corneal KSPGs and of neural crest/fibroblast differentiation into keratocytes. Specific Aims are to: 1) Determine if alternative splicing occurs during biosynthesis of the 3 KSPG core proteins in corneal keratocytes and in other fibroblastic and connective tissue cells in the eye and head regions; 2) Identify regulatory elements in genomic DNA that control biosynthesis of KSPGs in corneal keratocytes; 3) Identify factors that regulate glycosylation and sulfation of the three KSPG core proteins in the cornea and other tissues; 4) Characterize the expression patterns of the KSPG mRNAs and core proteins during corneal development, particularly in relation to timing and pattern of corneal innervation; 5) Determine the origin of corneal nerves - neural crest vs. ectodermal placode; 6) Determine the effect on corneal development and innervation of perturbing the biosynthesis of 1, 2, or all 3 corneal KSPGs; and 7) Determine if receptors for KSPGs occur on corneal cell types, as demonstrated on macrophages. Molecular biology methods will be applied to tissues from adult bovine eyes, as well as from those of quail and chicken embryos. Standard biochemical methods for proteoglycans, sugars, and sulfate ions will be used to focus on KSPGs as sensitive markers of keratocyte differentiation. Although transplanted corneas generally remain transparent re-innervation of the graft is very slow (years) and incomplete, depriving patients of corneal touch sensitivity. Research proposed may elucidate a relationship between KSPG distribution and pathways chosen by corneal nerves that mainly arise from the trigeminal ganglion.